Thousands of long noncoding (lnc) RNAs are induced with differentiation of human embryonic stem cells (hESCs) and are likely to control many stages of normal human development, but the functions of the vast majority of these noncoding genes remain unknown. The long term goal of this work is to determine the role of lncRNAs in endodermal differentiation and use this insight to develop new approaches to direct more efficient production of human tissues derived from endoderm. The overall objective of this proposal is to identify the functions of two lncRNAs that are induced during differentiation of hESCs into endoderm and in doing so to develop genetic tools to more efficiently assess the function of any lncRNA during endodermal differentiation. TGF- signaling is the key mediator of endodermal differentiation. The central hypothesis is that the lncRNAs that share promoter regions with developmental regulators and are activated by TGF- signaling are required for normal endodermal differentiation. Once the functions of key lncRNAs are understood in endodermal differentiation, it will then be possible to modulate expression of these lncRNAs to more efficiently control differentiation of tissue for regenerative medicine. The aims of this proposal are to determine the functions of the lncRNAs that are divergently transcribed from ID1 (lnc-ID1) and GSC (lnc-GSC) during endodermal differentiation. The working hypothesis is that both lnc-ID1, and lnc-GSC are required for normal endodermal differentiation. This hypothesis is based on the preliminary data that lnc-ID1 and lnc-GSC are induced by TGF- signaling and are coordinately regulated with the genes encoding ID1 and GSC, respectively, which are key proteins in hESC differentiation. Genome editing will be used to insert sequences to truncate lncRNA transcription in order to determine the requirement for each lncRNA in endodermal differentiation. Genome editing will also be used to create lncRNA-inducible hESC lines in order to assess the effect of a gain of function of each lncRNA on hESC state and endodermal differentiation. The contribution of this proposal is expected to be the determination of the roles of two lncRNAs in endodermal differentiation. This contribution is significant because the identification of lncRNAs that regulate endodermal differentiation will lead to more efficient production of human endodermal cells that can be used as a substrate for production of pancreatic, liver or intestinal cells for therapeutics. In addition, the genetic tools developed in this project will greatly facilitate analysis of the function of any lncRNA in human endodermal differentiation. The research proposed in this application is innovative because it represents a new and significant shift from current strategies focused on understanding how changes in expression of protein coding genes regulates endodermal differentiation and instead investigates how lncRNAs regulate the differentiation process.